β1 integrin is required for clustering and continued internalization of VEGFR2. (A) Internalization of VEGFR2 was analyzed in confluent HUVECs pretreated with β1 inhibitory antibodies or Ig control for 15 min, and then treated with Vs (200 ng/ml), Vb (200 ng/ml), and vehicle controls for another 15 min. To detect the internalized receptor, cells were treated with a recombinant single-chain antibody against human VEGFR2 (scFvA7), subsequently exposed to the indicated treatment followed by acid washes before lysis. Internalized VEGFR2 was analyzed by Western blot using VEGFR2 or 4G10 antibodies. Total VEGFR2 was determined in the same lysates. The numbers indicate densitometric analysis comparing Vs and Vb. (B) HUVECs pretreated with β1 inhibitory antibodies or Ig control for 15 min were exposed to vehicle (−), Vs (200 ng/ml), Vb (200 ng/ml), or collagen (col) for 15 min. Cells were fixed and stained using recombinant single-chain antibody against human VEGFR2 (scFvA7) with E-tag in the absence of permeabilization. FITC-conjugated anti–E-tag was used for detection of positive staining. Nuclei stained with TOPRO3 appears in blue. (C) Schematic model depicting the interaction of soluble and matrix-bound VEGF with VEGFR2. Soluble VEGF (left) results in rapid binding, internalization, and activation of VEGFR2. Matrix-bound VEGF (right) interacts easily with receptors as the cells migrate into the matrix. The affinity for the matrix is two orders of magnitude lower than its receptor. Over time, the progressive recruitment of integrins facilitates clustering of VEGFR2 and results in binding between integrins and VEGFR2. This interaction maintains the phosphorylation status of Y1214.